PROJECT SUMMARY The opioid epidemic in the United States is profound, with an annual economic burden of $95.8 billion dollars (2016 dollars) and a total cost of over $1Trillion since 2001. Moreover, drug overdose is now the no. 1 cause of accidental death with over 72,000 lives lost in 2017, and opioid overdoses account for over 60% of these deaths. In order to develop pharmacological and behavioral therapies to treat opioid use disorder (OUD), it is important to understand the neural circuitry and neuroadaptation that occurs following opioid use and withdrawal. Disorders on the affective spectrum often exhibit high comorbidity. Therefore, it is imperative to understand how opioids alter critical circuits and neurotransmitters that regulate addiction-like behaviors, aversion/anxiety and the response to stress. A node in the extended amygdala, the bed nucleus of the stria terminalis (BNST), receives the densest innervation of norepinephrine (NE, a stress responsive neurotransmitter) in the brain. The BNST is a major contributor to opioid withdrawal behaviors and previously, we demonstrated that morphine exposure and withdrawal modulates BNST NE release and uptake mechanisms in rats. Our preliminary data demonstrate that chronic stress enhances the same noradrenergic circuitry mice, and that opioid exposure and withdrawal modulates NE neurons. Furthermore, in mice we observe sex specific acute withdrawal behaviors, withdrawal induced disruption of sleep rhythms, and anxiety-like behavior in protracted abstinence. The BNST is a sexually dimorphic brain region, and we observe further sex differences in (and some similarities) in BNST physiology. Intriguingly, our data suggest that there may be reductions in excitatory transmission in select circuitry following opioid withdrawal, which may ultimately alter BNST output to classical reward circuits. These data inform the central hypothesis of our proposal investigated in 3 aims: opioid withdrawal 1. enhances the synaptic drive onto NE neurons innervating the BNST, 2. facilitates enhanced noradrenergic transmission within the BNST, and 3. induces glutamatergic plasticity within the BNST intensifying opioid withdrawal syndrome related behaviors.